Study animals and management

Records of 187 bulls (Holstein Frisian = 114, Jersey = 35, Borena = 12, 50%HF × 50%Borena = 5, 75%HF × 25%Borena = 15 and Fogera = 6) were considered and a total of 16,827 ejaculates (HF = 11172, Jersey = 2955, Borena = 424, 50%HF × 50%Borena = 690, 75%HF × 25%Borena = 1473 and Fogera = 113) collected from these breeding bulls for fifteen years (2005 - 2019) at the National Animal Genetic Improvement Institute (NAGII) were used in this study. The information recorded were bull identification number, breed, date of birth, date of collection, ejaculate volume (ml), semen colour, mass activity (scale: 1-4), concentration (10⁹ spz/ml), percentage of motile sperm at fresh, pre freeze and post freeze of 0 and 24 h and production doses/ejaculate. The bulls were kept indoor and under identical conditions of management, feeding and watering. They received hay, green forage and concentrate fortified with vitamins and minerals. Water was given ad libitum. They were allowed to exercise on running track weekly. Teaser bull was used during the collection of semen using Artificial Vagina (AV).

Age of the bulls were classified into four age classes (A1, < 2 years; A2, ≥ 2 to < 4 years; A3, ≥ 4 to < 6 years and A4, ≥ 6 years) and the factor production period (years of semen collection) was grouped into five (period-1=2005-2007, period-2=2008-2010, period-3=2011-2013, period-4=2014-2016 and period-5=2017-2019).

Semen preparation and evaluation

Semen was collected in the morning once a week from the bulls using sterilized artificial vagina (IMV; France) maintained at 45°C. Immediately after collection, volume of the ejaculate was measured with the aid of graduated collection tube. Colour of individual bull semen was evaluated by visual appraisal and recorded as creamy, milky, yellowish and watery. Sperm concentration was determined by using spectro photometer method. Evaluations of mass activity and individual motility were performed as described by Hafez (1993). Mass activity assessment was conducted by putting 5 µl of undiluted semen on pre warmed slide and examining it with stage warmed microscope at 37°C using a low magnification (100x). It was scored into 1 - 4 scales: 1= weak motion without forming any wave; 2= small, slow moving wave; 3= vigorous movement with moderate rapid waves and eddies and 4= dense, very rapidly moving waves and eddies. Fresh individual motility was estimated by placing 5 µl of undiluted semen on pre warmed slide and covering it with pre warmed coverslip (37°C) as percentage of spermatozoa. The sperm  cells  exhibiting  progressive  movements under stage warmed (37°C) phase contrast microscope at a magnification of 200X scored 0 to 100%. After the approval for further process, the extended semen was filled and sealed into 0.25 ml straws that were marked with the proper identification, and the straws were allowed to lay on ranks placed in a stabilizer-refrigerator at +4°C for 4 h of equilibration. After which the semen straws were transferred to a bio-freezer that gradually cools the semen to -140°C. Finally, before the semen is plunged into liquid nitrogen for long-term storage, post freezing individual motility was checked by randomly thawing 0.25 ml frozen semen straw of each bull at 37°C for 30 s in a water bath.

Post freezing individual motility evaluation was conducted at 0 and 24 h. Any sample showing below 40% individual motility was discarded. The semen straws passing this evaluation were kept in goblets and stored in liquid nitrogen at -196°C until dispatched for AI.

Statistical analysis

The data obtained were entered to Microsoft Excel sheet and Statistical Package for Social Science (SPSS) for windows version 20 (SPSS, 2011) was used for analysis. The data of ejaculate volume, concentration, mass activity, individual motility and Production doses/ejaculate were analysed using General Linear Model (GLM) for factorial experiments. Duncan’s Multiple Range Test (DMRT) and Chi square test were used to make specific comparison for significantly different fixed effects (breed, age and period of production) at a probability level of 5%. In the analysis, p ≤ 0.05 was set for level of significance. To evaluate the effect of breed, age and period of production on semen quality traits, the following model was fitted for analyses.

Yijk = μ + Bi + Aj + Pk + eijk

where Yijk - observed semen characteristics; µ - overall mean; B_i - fixed effect of the i^th breed (i=1- 6); Aj - fixed effect of the j^th age class (j=1- 4); P_l - fixed effect of the l^th period at collection (l=1- 5); and e_ijk - a random residual effect.

Effect of breed

As illustrated in Table 1 significant (p ≤ 0.05) differences for ejaculate volume, concentration, mass activity, individual motility and production doses/ejaculate were recorded among breeds. All these semen quality measures were significantly (p ≤ 0.05) minimal in 50%HF × 50%Borena crosses. Significantly (p ≤ 0.05) higher ejaculate volume was recorded in HF and 75%HF × 25%Borena crosses. In line with this, significantly (p ≤ 0.05) higher production doses of semen were recorded in HF.

In contrast, significantly (p ≤ 0.05) higher sperm concentration was detected in Borena semen.  Significantly (p ≤ 0.05) higher mass activity and fresh individual motility percentage were observed in local zebu (Borena and Fogera) and exotic (HF and Jersey) breeds as compared to cross breeds. However, post freezing individual motilities at 0 and 24 h were significantly (p ≤ 0.05) higher for exotic (HF and Jersey) breeds.

Effect of age

The present study demonstrates that age had significant (p ≤ 0.05) effect on all semen quality measures: ejaculate volume, concentration, mass activity, individual motilities of all stages of production and production doses/ejaculate (Table 2).

In this study, breed affected significantly (p ≤ 0.05) semen ejaculate volume, concentration, mass activity, individual motilities and production doses/ejaculate. These semen quality measures were significantly (p ≤ 0.05) minimal in 50%HF × 50%Borena crosses.  Although significant differences among certain breeds for some semen quality measures were not recorded, all semen quality measures except concentration were observed superior in HF breed. Breed differences on several semen parameters have been previously reported (Hafez, 1993; Brito et al., 2002). As it was stated by Bang (2008), Holstein Friesian breed is known to give best results compared to other breeds which were also in close agreement with the current findings. Previously, different authors (Hunderra, 2004; Sinishaw, 2005; Desalegn  et  al.,  2009;  Demeke,  2010;  Lemma,  2011; Lemma and Shemsu, (2015) have also reported the semen characteristics difference among cattle breeds at the same AI center. But the finding of Belayneh (2018) did not state this breed difference which might be due to short-term study period and a smaller number of observations in his study.

Like breed, age had also significant (p ≤ 0.05) effect on all semen quality measures: ejaculate volume, concentration, mass activity, individual motilities and production doses/ejaculate. Except ejaculate volume and production doses, all the semen quality measures were recorded better in the first two age classes (less than 2 and 2 to 4 years of age classes). Volume and production doses per ejaculate tend to increase with age of the bull up to the third class of age (6 years). Better semen quality in mature bulls compared to younger and older bulls may probably be due to scrotal circumference and the heat regulation mechanisms, which increases with age until the rate of broken down for testicular tissues become faster than being replaced (King, 1993; Brito et al., 2002). Moreover, effect of age is also influenced by managemental conditions. As artificial insemination centers are highly interested in producing a maximum number of straws, less demanded bulls might not reach at a higher age. Therefore, it can be emphasized that bull management in the younger age also had significant effect in quality semen productivity. The effect of age also attributed to fat deposition in the scrotum, which increases with age and can lower the efficiency of scrotal thermo-regulation by reducing the amount of heat that can be radiated from the scrotal neck; and hence inferior semen quality will be produced in old bulls (Barth and Oko, 1989; Coulter et al., 1997; Kommisrud and Berg, 1996). Time of sexual preparation also have significant effects on ejaculate volume and number of doses per ejaculate. This  finding is in close agreement with the findings of Hunderra (2004), Sinishaw (2005), Desalegn et al. (2009), Demeke (2010), Lemma (2011), Lemma and Shemsu (2015).

In this study period of production had also significant (p ≤ 0.05) effect on ejaculate volume, mass activity, all stages of individual motilities and production doses/ejaculate. The significant difference of ejaculate volume, concentration, mass activity, and individual motilities among different production periods might be due to variation in managemental practice and efficiency of implementing the protocol in selecting candidate bulls for the time period.

The results of this study clearly demonstrated that breed, age and period of semen production have significant effect on bovine semen characteristics. Holstein Frisian semen was best almost for all semen quality measures followed by Jersey; on the other hand, semen quality of cross breed bulls (in  particular  50%  HF ×  50% Borena) was significantly inferior (P < 0.05) in all semen quality measures. The mid two age classes (matured bulls up to 6 years of old) were able to produce better quality semen which might be due to better testicular and preferential managemental conditions. Therefore, to produce good quality bovine semen, NAGII shall work tough giving emphasis for breeds of HF and Jersey, matured bulls up to 6 years of age.

The authors have not declared any conflict of interests.

The authors thank National Animal Genetic Improvement Institute, in particular Dr. Esayase Tesema Deputy General Director, Dr. Bula Agegnehu Cattle Genetic Improvement Recording and feedback Directorate Director for their willingness to use the data and W/rt Wineshet for her committed and precise data management and handling using sperm software.